PKC-theta is a component of the immunological synapse that plays an essential role in mature T cell activation by integrating T cell receptor (TCR)/CD28 costimulatory signals that activate transcription factors NF-kappaB and AP-1. These events are defective in anergic T cells. This application is based on the following hypotheses: a) Proper function of PKC-theta in antigen-specific T cells depends upon its selective localization to the T cell synapse and is important for preventing T cell anergy. b) A scaffold protein links PKC-theta to specific membrane and/or cytoskeleton compartments and selectively recruits it to the T cell synapse. The following Aims will address these hypotheses: 1 ) We will map the critical residues/regions in the regulatory domain of PKC-theta that are responsible for its selective translocation to the T cell synapse and co-localized lipid rafts. 2) We will determine whether either one of two candidate PDZ proteins, hDLG or p55, represents the PKC-theta-selective scaffold and, if not, embark on a broad search to isolate the putative PKC-theta scaffold. We will then conduct a detailed structure-function analysis to determine the functional significance of the interaction between PKC-theta and its scaffold in antigen-induced T cell activation. 3) We will establish T cell lines expressing beta-lactamase-based AP-1 and CD28RE reporter genes. These lines will be used as sensitive biosensors in a high throughput screen of compound libraries in order to isolate and functionally characterize compounds that selectively interfere with PKC-theta function and its selective recruitment to the T cell synapse. The proposed studies directly address one goal of this RFA, i.e., "identification and characterization of promising new T or B cell molecular targets for tolerance induction". They offer the potential for developing novel therapeutic strategies, which may be useful for modulating CD28 costimulation in disease.